Alkaline shock-based preparation of nucleic acids

ABSTRACT

Method of preparing a biological sample appropriate for use in a subsequent in vitro nucleic acid amplification reaction. The method involves a rapid, transient exposure to alkaline conditions which can be achieved by mixing an alkaline solution with a pH-buffered solution that includes a detergent and the biological sample to be tested for the presence of particular nucleic acid species using in vitro amplification. The invented method advantageously can improve detection of some target nucleic acids without substantially compromising detectability of others. The method is particularly useful for simultaneously preparing RNA and DNA templates that can be used in multiplex amplification reactions.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/356,613, now U.S. Pat. No. 7,510,837, which claims the benefit ofU.S. Provisional Application No. 60/654,199, filed Feb. 18, 2005, andU.S. Provisional Application No. 60/669,192, filed Apr. 6, 2005. Thedisclosures of these prior applications are hereby incorporated byreference.

FIELD OF THE INVENTION

The present invention relates to nucleic acid amplification technology.More specifically, the invention relates to a method of preparing asample preliminary to conducting an in vitro nucleic acid amplificationreaction.

BACKGROUND OF THE INVENTION

In vitro nucleic acid amplification techniques are now commonly used forsynthesizing, and perhaps detecting vanishingly small quantities of anucleic acid target. These techniques conventionally employ one or moreoligonucleotide primers and a nucleic acid-polymerizing enzyme tosynthesize copies of one or both strands of a nucleic acid template.Many different methods have been used for preparing biological samplesin advance of the amplification procedure.

Multiplexed assays, which are capable of amplifying any of a pluralityof different nucleic acid targets from a test sample in a singlereaction, present special design challenges. For example, the targetsamplifiable in a multiplex assay may be RNA targets, DNA targets, oreven a combination of RNA and DNA targets. One challenge arises from thefact that RNA and DNA nucleic acids exhibit different chemicalstabilities. Another challenge arises from a common desire to detect,with maximum sensitivity, any of a variety of related subtypes of asingle target species. Even when subtype-specific primers are used inthe reactions, it can be difficult to achieve substantially similardetection sensitivity for different subtypes of a single type oforganism.

Accordingly, there is a need for a general technique which can enhancedetectability of particular targets in nucleic acid amplificationreactions. There is a further need for enhancing detectability of one ormore targets in multiplex amplification reactions without substantiallysacrificing detectability of other targets in the same reaction. Thepresent invention addresses these needs.

Indeed, the invention disclosed herein provides a convenient method forpreparing biological samples to be tested for the presence of nucleicacid targets using in vitro nucleic acid amplification. This methodadvantageously provides reliable results with a variety of nucleicacid-containing biological samples, while dramatically improvingdetectability of certain nucleic acid targets.

SUMMARY OF THE INVENTION

General speaking, the invention relates to a method of processing abiological sample. This method begins with a step for combining thebiological sample with a pH buffer and a detergent to result in a firstliquid composition having a first pH. It is convenient for the pH bufferand for the detergent to be in liquid form to simplify the combiningstep. Next, there is a step for mixing with the first liquid compositionan alkaline composition to result in a second liquid composition havinga second pH. Importantly, the second pH must be at least 0.2 pH unitshigher than the first pH, this being due to the added alkali. It is alsoimportant for the second pH to be lower than pH 9.5 to achieve goodresults. This is followed by a step for capturing one or more nucleicacids from the second liquid composition onto a solid support. Finally,there is a step for isolating the solid support having captured thereonany of the one or more nucleic acids. This may, for example, involveaspirating non-bound materials that remain in the liquid phase, therebyphysically isolating the solid support and any nucleic acids capturedthereon.

In one embodiment, the pH buffer and the detergent used in the combiningstep are each components of a buffered detergent solution, and thecombining step involves combining the biological sample with an aliquotof the buffered detergent solution.

In another embodiment, the second pH, meaning the pH of the mixture thatincludes the biological sample, the buffer and the alkaline composition,is in the range of from pH 8.0 to pH 9.2. Preferably, when the second pHfalls in the range of from pH 8.0 to 9.2, the capturing step involvescapturing one or more RNA species or capturing one or more DNA speciesonto the solid support. In another preferred embodiment, when the secondpH falls in the range of from pH 8.0 to 9.2, the first pH is in therange of from 6.5 to 8.0. In yet another preferred embodiment, when thesecond pH falls in the range of from pH 8.0 to 9.2, the capturing stepcan involve hybridizing one or more nucleic acids from the second liquidcomposition to one or more immobilized or immobilizable oligonucleotidescomplementary thereto. More preferably, the pH buffer and the detergentin the combining step are each components of a buffered detergentsolution, and the combining step involves combining the biologicalsample with an aliquot of the buffered detergent solution. Still morepreferably, the buffered detergent solution which includes the pH bufferand the detergent further includes the immobilized or immobilizableoligonucleotides that can be used for hybridizing and capturing nucleicacids from the second liquid composition that resulted from the mixingstep.

In another preferred embodiment, the first pH, meaning the pH of thecombination of the biological sample, the buffer and the detergent, isin the range of from 6.5 to 8.0. When this is the case, the capturingstep preferably involves hybridizing the one or more nucleic acids to becaptured to one or more immobilized or immobilizable oligonucleotidescomplementary thereto. More preferably, the pH buffer and the detergentused in the combining step are each components of a buffered detergentsolution, and the combining step involves combining the biologicalsample with an aliquot of the buffered detergent solution. Still morepreferably, the buffered detergent solution further includes the one ormore immobilized or immobilizable oligonucleotides that are used forcapturing nucleic acids from the second liquid composition. Yet stillmore preferably, the isolating step involves separating the solidsupport from material not captured thereon, and then washing the solidsupport having captured thereon any nucleic acids. Even yet still morepreferably, all of the above-described steps are carried out in a singlereaction vessel.

In another preferred embodiment, the capturing step involves hybridizingthe one or more nucleic acids that are to be captured to one or moreimmobilized or immobilizable oligonucleotides complementary thereto.When this is the case, it is preferred for the pH buffer and thedetergent used in the combining step to each be components of a buffereddetergent solution, and for the combining step to involve combining thebiological sample with an aliquot of the buffered detergent solution.More preferably, this buffered detergent solution further includes theone or more immobilized or immobilizable oligonucleotides that can beused for capturing nucleic acids from the second liquid composition.

In another preferred embodiment, the detergent used in the combiningstep is an anionic detergent or a non-ionic detergent. When this is thecase, the alkaline composition used in the mixing step preferably is astrong base, such as NaOH or LiOH.

In another embodiment, all of the steps for combining, mixing, capturingand isolating are carried out in a single reaction vessel. When this isthe case, the mixing step preferably involves either agitating byorbital shaking or vortexing.

In another embodiment, the solid support in the capturing step includesa bead, such as a magnetic bead.

In another embodiment, the pKa of the pH buffer used in the combiningstep is between 6.0 and 9.0.

In another embodiment, at least one of the one or more nucleic acidscaptured in the capturing step is an RNA molecule.

In another embodiment, at least one of the one or more nucleic acidscaptured in the capturing step is a DNA molecule. In another preferredembodiment, the first pH falls in the range of from pH 6.5 to 8.0, andthe second pH falls in the range of from pH 8.2 to 9.2. Thisrelationship gave universally good results for processing both DNAtemplates and RNA templates. Thus, this combination of ranges is highlypreferred for carrying out the invention.

Another general aspect of the invention relates to a method ofprocessing a biological sample to obtain nucleic acids, and then usingthe obtained nucleic acids in a particular application. As above, thismethod begins with a step for combining the biological sample with a pHbuffer and a detergent to result in a first liquid composition having afirst pH. Next, there is a step for mixing with the first liquidcomposition an alkaline composition to result in a second liquidcomposition having a second pH. Again, it is important for the second pHto be at least 0.2 pH units higher than the first pH, and for the secondpH to be lower than pH 9.5 to achieve good results. This is followed bya step for capturing one or more nucleic acids from the second liquidcomposition onto a solid support. Next, there is a step for isolatingthe solid support having captured thereon any of the one or more nucleicacids. This may, for example, involve aspirating non-bound materialsthat remain in the liquid phase, thereby physically isolating the solidsupport and any nucleic acids captured thereon. Finally, there is a stepfor performing an in vitro nucleic acid amplification reaction using asa template at least one of the nucleic acids captured on the solidsupport and isolated in the isolating step.

In a preferred embodiment, the second pH is in the range of from pH 8.0to pH 9.2. When this is the case, it is preferred for the first pH to bein the range of from 6.5 to 8.0.

In another preferred embodiment, the first pH is in the range of from6.5 to 8.0. When the first pH falls in the range of from 6.5 to 8.0, itis highly desirable for the second pH to fall in the range of from pH8.2 to 9.2. Indeed, this set of ranges gave universally good results forprocessing both DNA templates and RNA templates. Thus, this combinationof ranges is highly preferred for carrying out the invention.

In a different preferred embodiment, the in vitro nucleic acidamplification reaction is a multiplex reaction capable of amplifyingmore than one nucleic acid sequence. When this is the case, the one ormore nucleic acids captured in the capturing step include two or morecaptured nucleic acids; the one or more nucleic acids isolated in theisolating step include two or more isolated nucleic acids; and themultiplex reaction uses as templates two of the of the captured andisolated nucleic acids. Indeed, it is highly preferred for the twonucleic acids used as templates in the multiplex reaction to include anRNA molecule and a DNA molecule.

In yet a different preferred embodiment, all of the steps for combining,mixing, capturing, isolating, and performing the nucleic acidamplification reaction are carried out in a single reaction vessel. Whenthis is the case, it is preferred for the mixing step to involve eitheragitating by orbital shaking or vortexing.

DEFINITIONS

The following terms have the following meanings for the purpose of thisdisclosure, unless expressly stated to the contrary herein.

As used herein, “alkaline shock” refers to a transient high pH effectedby first combining a biological sample with a pH buffer and a detergentto result in a first composition, and then mixing with that firstcomposition an amount of an alkaline composition sufficient to increasethe pH of the resulting mixture. Useful starting ranges for the pH ofthe first composition, and useful final ranges for the pH of the mixturesubsequent to the addition of the alkaline composition are describedherein.

As used herein, a “biological sample” is any tissue orpolynucleotide-containing material obtained from a human, animal orenvironmental sample. Biological samples in accordance with theinvention include peripheral blood, plasma, serum or other body fluid,bone marrow or other organ, biopsy tissues or other materials ofbiological origin. A biological sample may be treated to disrupt tissueor cell structure, thereby releasing intracellular components into asolution which may contain enzymes, buffers, salts, detergents and thelike.

As used herein, “polynucleotide” means either RNA or DNA, along with anysynthetic nucleotide analogs or other molecules that may be present inthe sequence and that do not prevent hybridization of the polynucleotidewith a second molecule having a complementary sequence.

As used herein, a “detectable label” is a chemical species that can bedetected or can lead to a detectable response. Detectable labels inaccordance with the invention can be linked to polynucleotide probeseither directly or indirectly, and include radioisotopes, enzymes,haptens, chromophores such as dyes or particles that impart a detectablecolor (e.g., latex beads or metal particles), luminescent compounds(e.g., bioluminescent, phosphorescent or chemiluminescent moieties) andfluorescent compounds.

A “homogeneous detectable label” refers to a label that can be detectedin a homogeneous fashion by determining whether the label is on a probehybridized to a target sequence. That is, homogeneous detectable labelscan be detected without physically removing hybridized from unhybridizedforms of the label or labeled probe. Homogeneous detectable labels arepreferred when using labeled probes for detecting amplified nucleicacids. Examples of homogeneous labels have been described in detail byArnold et al., U.S. Pat. No. 5,283,174; Woodhead et al., U.S. Pat. No.5,656,207; and Nelson et al., U.S. Pat. No. 5,658,737. Preferred labelsfor use in homogenous assays include chemiluminescent compounds (e.g.,see Woodhead et al., U.S. Pat. No. 5,656,207; Nelson et al., U.S. Pat.No. 5,658,737; and Arnold, Jr., et al., U.S. Pat. No. 5,639,604).Preferred chemiluminescent labels are acridinium ester (“AE”) compounds,such as standard AE or derivatives thereof (e.g., naphthyl-AE, ortho-AE,1- or 3-methyl-AE, 2,7-dimethyl-AE, 4,5-dimethyl-AE, ortho-dibromo-AE,ortho-dimethyl-AE, meta-dimethyl-AE, ortho-methoxy-AE,ortho-methoxy(cinnamyl)-AE, ortho-methyl-AE, ortho-fluoro-AE, 1- or3-methyl-ortho-fluoro-AE, 1- or 3-methyl-meta-difluoro-AE, and2-methyl-AE).

A “homogeneous assay” refers to a detection procedure that does notrequire physical separation of hybridized probe from non-hybridizedprobe prior to determining the extent of specific probe hybridization.Exemplary homogeneous assays, such as those described herein, can employmolecular beacons or other self-reporting probes which emit fluorescentsignals when hybridized to an appropriate target, chemiluminescentacridinium ester labels which can be selectively destroyed by chemicalmeans unless present in a hybrid duplex, and other homogeneouslydetectable labels that will be familiar to those having an ordinarylevel of skill in the art.

As used herein, “nucleic acid amplification,” or simply “amplification”refers to an in vitro procedure for obtaining multiple copies of atarget nucleic acid sequence, its complement or fragments thereof.

By “target nucleic acid” or “target” is meant a nucleic acid containinga target nucleic acid sequence. In general, a target nucleic acidsequence that is to be amplified will be positioned between twooppositely disposed amplification oligonucleotides, and will include theportion of the target nucleic acid that is fully complementary to eachof the amplification oligonucleotides.

By “target nucleic acid sequence” or “target sequence” or “targetregion” is meant a specific deoxyribonucleotide or ribonucleotidesequence comprising all or part of the nucleotide sequence of asingle-stranded nucleic acid molecule, and the deoxyribonucleotide orribonucleotide sequence complementary thereto.

By “transcription associated amplification” is meant any type of nucleicacid amplification that uses an RNA polymerase to produce multiple RNAtranscripts from a nucleic acid template. One example of a transcriptionassociated amplification method, called “Transcription MediatedAmplification” (TMA), generally employs an RNA polymerase, a DNApolymerase, deoxyribonucleoside triphosphates, ribonucleosidetriphosphates, and a promoter-template complementary oligonucleotide,and optionally may include one or more analogous oligonucleotides.Variations of TMA are well known in the art as disclosed in detail inBurg et al., U.S. Pat. No. 5,437,990; Kacian et al., U.S. Pat. Nos.5,399,491 and 5,554,516; Kacian et al., PCT No. WO 93/22461; Gingeras etal., PCT No. WO 88/01302; Gingeras et al., PCT No. WO 88/10315; Malek etal., U.S. Pat. No. 5,130,238; Urdea et al., U.S. Pat. Nos. 4,868,105 and5,124,246; McDonough et al., PCT No. WO 94/03472; and Ryder et al., PCTNo. WO 95/03430. The methods of Kacian et al. are preferred forconducting nucleic acid amplification procedures of the type disclosedherein.

As used herein, an “oligonucleotide” or “oligomer” is a polymeric chainof at least two, generally between about five and about 100, chemicalsubunits, each subunit comprising a nucleotide base moiety, a sugarmoiety, and a linking moiety that joins the subunits in a linear spacialconfiguration. Common nucleotide base moieties are guanine (G), adenine(A), cytosine (C), thymine (T) and uracil (U), although other rare ormodified nucleotide bases able to hydrogen bond are well known to thoseskilled in the art. Oligonucleotides may optionally include analogs ofany of the sugar moieties, the base moieties, and the backboneconstituents. Preferred oligonucleotides of the present invention fallin a size range of about 10 to about 100 residues. Oligonucleotides maybe purified from naturally occurring sources, but preferably aresynthesized using any of a variety of well known enzymatic or chemicalmethods.

As used herein, a “probe” is an oligonucleotide that hybridizesspecifically to a target sequence in a nucleic acid, preferably in anamplified nucleic acid, under conditions that promote hybridization, toform a detectable hybrid. A probe optionally may contain a detectablemoiety which either may be attached to the end(s) of the probe or may beinternal. The nucleotides of the probe which combine with the targetpolynucleotide need not be strictly contiguous, as may be the case witha detectable moiety internal to the sequence of the probe. Detection mayeither be direct (i.e., resulting from a probe hybridizing directly tothe target sequence or amplified nucleic acid) or indirect (i.e.,resulting from a probe hybridizing to an intermediate molecularstructure that links the probe to the target sequence or amplifiednucleic acid). The “target” of a probe generally refers to a sequencecontained within an amplified nucleic acid sequence which hybridizesspecifically to at least a portion of a probe oligonucleotide usingstandard hydrogen bonding (i.e., base pairing). A probe may comprisetarget-specific sequences and optionally other sequences that arenon-complementary to the target sequence that is to be detected. Thesenon-complementary sequences may comprise a promoter sequence, arestriction endonuclease recognition site, or sequences that contributeto three-dimensional conformation of the probe (e.g., as described inLizardi et al., U.S. Pat. Nos. 5,118,801 and 5,312,728). Sequences thatare “sufficiently complementary” allow stable hybridization of a probeoligonucleotide to a target sequence that is not completelycomplementary to the probe's target-specific sequence.

By “amplification oligonucleotide” is meant an oligonucleotide that iscapable of participating in a nucleic acid amplification reaction tobring about the synthesis of multiple copies of a template nucleic acidsequence, or its complement. It is common for amplification reactions toemploy at least two amplification oligonucleotides, with at least one ofthe amplification oligonucleotides serving as an amplification primer.

As used herein, an “amplification primer,” or more simply “primer,” isan oligonucleotide that hybridizes to a target nucleic acid, or itscomplement, and can be extended in a template-dependent primer extensionreaction. For example, amplification primers may be optionally modifiedoligonucleotides which are capable of hybridizing to a template nucleicacid, and which have a 3′ end that can be extended by a DNA polymeraseactivity. In general, a primer will have a downstreamtarget-complementary sequence, and optionally an upstream sequence thatis not complementary to target nucleic acids. The optional upstreamsequence may, for example, serve as an RNA polymerase promoter orcontain restriction endonuclease cleavage sites.

By “substantially homologous,” “substantially corresponding” or“substantially corresponds” is meant that the subject oligonucleotidehas a base sequence containing an at least 10 contiguous base regionthat is at least 70% homologous, preferably at least 80% homologous,more preferably at least 90% homologous, and most preferably 100%homologous to an at least 10 contiguous base region present in areference base sequence (excluding RNA and DNA equivalents). Thoseskilled in the art will readily appreciate modifications that could bemade to the hybridization assay conditions at various percentages ofhomology to permit hybridization of the oligonucleotide to the targetsequence while preventing unacceptable levels of non-specifichybridization. The degree of similarity is determined by comparing theorder of nucleobases making up the two sequences and does not take intoconsideration other structural differences which may exist between thetwo sequences, provided the structural differences do not preventhydrogen bonding with complementary bases. The degree of homologybetween two sequences can also be expressed in terms of the number ofbase mismatches present in each set of at least 10 contiguous basesbeing compared, which may range from 0-2 base differences.

By “substantially complementary” is meant that the subjectoligonucleotide has a base sequence containing an at least 10 contiguousbase region that is at least 70% complementary, preferably at least 80%complementary, more preferably at least 90% complementary, and mostpreferably 100% complementary to an at least 10 contiguous base regionpresent in a target nucleic acid sequence (excluding RNA and DNAequivalents). (Those skilled in the art will readily appreciatemodifications that could be made to the hybridization assay conditionsat various percentages of complementarity to permit hybridization of theoligonucleotide to the target sequence while preventing unacceptablelevels of non-specific hybridization.) The degree of complementarity isdetermined by comparing the order of nucleobases making up the twosequences and does not take into consideration other structuraldifferences which may exist between the two sequences, provided thestructural differences do not prevent hydrogen bonding withcomplementary bases. The degree of complementarity between two sequencescan also be expressed in terms of the number of base mismatches presentin each set of at least 10 contiguous bases being compared, which mayrange from 0-2 base mismatches.

By “sufficiently complementary” is meant a contiguous nucleic acid basesequence that is capable of hybridizing to another base sequence byhydrogen bonding between a series of complementary bases. Complementarybase sequences may be complementary at each position in the basesequence of an oligonucleotide using standard base pairing (e.g., G:C,A:T or A:U pairing) or may contain one or more residues that are notcomplementary using standard hydrogen bonding (including abasic“nucleotides”), but in which the entire complementary base sequence iscapable of specifically hybridizing with another base sequence underappropriate hybridization conditions. Contiguous bases are preferably atleast about 80%, more preferably at least about 90%, and most preferablyabout 100% complementary to a sequence to which an oligonucleotide isintended to specifically hybridize. Appropriate hybridization conditionsare well known to those skilled in the art, can be predicted readilybased on base sequence composition, or can be determined empirically byusing routine testing (e.g., See Sambrook et al., Molecular Cloning, ALaboratory Manual, 2^(nd) ed. (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989) at §§ 1.90-1.91, 7.37-7.57, 9.47-9.51 and11.47-11.57 particularly at §§ 9.50-9.51, 11.12-11.13, 11.45-11.47 and11.55-11.57).

By “capture oligonucleotide” is meant at least one nucleic acidoligonucleotide that provides means for specifically joining a targetsequence and an immobilized oligonucleotide due to base pairhybridization. A capture oligonucleotide preferably includes two bindingregions: a target sequence-binding region and an immobilizedprobe-binding region, usually contiguous on the same oligonucleotide,although the capture oligonucleotide may include a targetsequence-binding region and an immobilized probe-binding region whichare present on two different oligonucleotides joined together by one ormore linkers. For example, an immobilized probe-binding region may bepresent on a first oligonucleotide, the target sequence-binding regionmay be present on a second oligonucleotide, and the two differentoligonucleotides are joined by hydrogen bonding with a linker that is athird oligonucleotide containing sequences that hybridize specificallyto the sequences of the first and second oligonucleotides.

By “immobilized oligonucleotide” or “immobilized nucleic acid,” andvariants thereof, is meant a nucleic acid that joins, directly orindirectly, a capture oligonucleotide to an immobilized support. Animmobilized probe is an oligonucleotide joined to a solid support thatfacilitates separation of bound target sequence from unbound material ina sample. An “immobilizable” oligonucleotide is an oligonucleotide thatcan, by way of complementary base interactions with an oligonucleotideimmobilized directly to a solid support, become immobilized to the solidsupport.

By “separating” or “purifying” is meant that one or more components ofthe biological sample are removed from one or more other components ofthe sample. Sample components include nucleic acids in a generallyaqueous solution phase which may also include materials such asproteins, carbohydrates, lipids and labeled probes. Preferably, theseparating or purifying step removes at least about 70%, more preferablyat least about 90% and, even more preferably, at least about 95% of theother components present in the sample.

By “RNA and DNA equivalents” or “RNA and DNA equivalent bases” is meantmolecules, such as RNA and DNA, having the same complementary base pairhybridization properties. RNA and DNA equivalents have different sugarmoieties (i.e., ribose versus deoxyribose) and may differ by thepresence of uracil in RNA and thymine in DNA. The differences betweenRNA and DNA equivalents do not contribute to differences in homologybecause the equivalents have the same degree of complementarity to aparticular sequence.

By “consisting essentially of” is meant that additional component(s),composition(s) or method step(s) that do not materially change the basicand novel characteristics of the present invention may be included inthe compositions or kits or methods of the present invention. Suchcharacteristics include the ability to selectively detect target nucleicacids in biological samples such as whole blood or plasma. Anycomponent(s), composition(s), or method step(s) that have a materialeffect on the basic and novel characteristics of the present inventionwould fall outside of this term.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram illustrating the various polynucleotidesthat can be used for detecting a target region within a model targetnucleic acid (represented by a thick horizontal line). Positions of thefollowing nucleic acids are shown relative to the target region:“Capture Oligonucleotide” refers to the nucleic acid used to hybridizeto and capture the target nucleic acid prior to amplification, where “T”refers to a tail sequence used to hybridize an immobilizedoligonucleotide having a complementary sequence (not shown); “Non-T7Primer” and “T7 Promoter-Primer” represent two amplification primersused for conducting TMA, where “P” indicates the promoter sequence ofthe T7 promoter-primer; and “Probe” refers to the probe used fordetecting amplified nucleic acid.

FIGS. 2A-2C are a series of bar graphs displaying results from trialsconducted using different concentrations of NaOH solution. FIG. 2Apresents results measured as % Positivity as a function of theconcentration of NaOH used in the alkaline shock step. FIG. 2B presentsresults measured as % CV (coefficient of variability) as a function ofthe concentration of NaOH used in the alkaline shock step. FIG. 2Cpresents results measured as the Mean RLU as a function of theconcentration of NaOH used in the alkaline shock step.

FIGS. 3A-3C are a series of bar graphs displaying results measured as %Reactive for different levels of input HBV virus. FIG. 3A shows resultsfor HBV subtype-B. FIG. 3B shows results for HBV subtype-C. FIG. 3Cshows results for HBV subtype-A. Control trials are indicated by openbars. Results from trials receiving an alkaline shock are indicated byfilled bars.

FIGS. 4A-4C are a series of bar graphs displaying results measured as %Reactive for different levels of input RNA viruses. FIG. 4A showsresults for HCV-1a virus. FIG. 4B shows results for HCV-2b virus. FIG.4C shows results for HIV-1b virus. Control trials are indicated by openbars. Results from trials receiving an alkaline shock are indicated byfilled bars.

FIG. 5 shows a series of bar graphs displaying % positivity determinedin amplification reactions using template nucleic acids isolated fromdifferent numbers of GBS bacteria. Trials employing nucleic acidtemplates processed without an alkaline shock are indicated by openbars. Trials employing nucleic acid templates processed with an alkalineshock are indicated by filled bars.

DETAILED DESCRIPTION OF THE INVENTION

Herein there is disclosed a method of preparing a nucleicacid-containing biological sample. The method can be used to prepareboth DNA and RNA templates from viral, bacterial or eucaryotic sources,and can be used for enhancing the sensitivity of amplification reactionsconducted using the prepared nucleic acids as templates. Surprisingly,all of these advantages can be achieved without redesigning any of theoligonucleotides used in the target-capture, amplification, or detectionsteps of a nucleic acid detection procedure.

Generally speaking, subject matter of the invention relates to theunexpected discovery that addition of an alkaline solution to a buffereddetergent solution which contains a biological sample, such that the pHafter mixing falls within a specified range, provides certain advantageswhen the processed sample subsequently serves as a source of templatesin a nucleic acid amplification reaction. Preferably, the inventedsample processing procedure involves a target-capture component whereinconditions following addition of the alkali are compatible with theformation of polynucleotide hybrids that include an immobilized captureoligonucleotide and a polynucleotide liberated from the biologicalsample. As indicated by the evidence presented below, advantages of theinvention are not achieved when the alkali and pH buffered detergentsolutions are first combined, and then added to the biological sample.

An observation which prompted development of the invention related tothe differential ability of an amplified assay to detect different HBVsubtypes. More specifically, a multiplexed assay capable of amplifyingand detecting any of HIV-1, HCV and HBV nucleic acid targets was foundto exhibit widely different sensitivities for different HBV subtypes. Asindicated below, the assay yielded approximately equivalentsensitivities for subtypes-A and-C, but a sensitivity for subtype-B thatwas nearly 16 fold reduced. This was despite the fact that the threesubtypes of HBV share a close phylogenetic relationship, and despite thefact that a common set of primers and probes can be used in the assayprocedure. Because it was desirable to detect all of the differentsubtypes with great sensitivity, it was of interest to enhance assaysensitivity for detection of one viral subtype without substantiallycompromising detection of the other subtypes.

Improving the sensitivity of HBV subtype-B detection conceivably couldhave been accomplished by any of several approaches. For example,redesigning the oligonucleotide components used in the assay may haveled to improvements. However, even if this could have been accomplished,the solution would only have been specific for the redesigned assay. Amore desirable solution would provide a general means for improving HBVsubtype-B detectability, and perhaps the performance of other assays aswell. Thus, one object of the invention related to a method of improvingthe detectability of at least one target in a multiplexed assay.

Preferred Buffers and pH Ranges

Buffers useful for carrying out the invented alkaline shock-based samplepreparation method preferably have pKa values in the range of from about6.0 to about 9.0. An exemplary buffer used for demonstrating utility ofthe invention is HEPES (N-2-Hydroxyethylpiperazine-N′-2-Ethane SulfonicAcid), which has a pKa of 7.55 at 20° C., and which has its strongestbuffer capacity in the pH range of from 6.8 to 8.2. Of course, successof the technique is not limited by the use of any particular buffer.

According to a preferred method for carrying out the invention, abiological sample is first combined with a pH buffer and a detergent togive a first composition, which is then combined with an aliquot of aconcentrated hydroxide solution to effect the alkaline shock. The bufferand detergent conveniently can be combined with each other so that asingle aliquot of a buffered detergent solution can be dispensed in areagent addition step. It is preferred for the buffered detergentsolution to additionally contain one or more optional immobilizable orimmobilized capture oligonucleotides to further reduce the complexity ofthe reagent addition steps, thereby particularly adapting the method toautomation by the use of robotic pipettors. When the alkaline shocksample preparation method is performed by combining an aliquot of aliquid, or liquified biological sample with a single reagent compositionthat contains the buffer, the detergent, and the capture oligonucleotideand a solid support (i.e., a bead) to effect the capture, the reagentcomposition is termed, “lysis/capture reagent.” Excellent results havebeen achieved using lysis/capture reagents having buffer concentrationsin the range of from about 200 mM to about 800 mM, which produced finalbuffer concentrations after combining lysis/capture reagent with thebiological sample in the range of from about 90 mM to about 355 mM. Ofcourse, changes to the buffer strength require that the amount of addedalkaline hydroxide be adjusted to bring the final pH of the mixture intoone of the ranges specified herein. Thus, success of the technique isnot limited by the amounts or concentrations of the buffer in themixture prior to addition of the alkaline solution which effectsalkaline shock.

Preferably the starting pH range for the combination of a buffereddetergent solution mixed with a biological sample before addition ofalkali to effect that alkaline shock is in the range of from pH 6.5 to8.0, still more preferably in the range of from pH 7.0 to 8.0, or yetstill more preferably in the range of from pH 7.0 to 7.5. Testingconducted using buffered detergent solutions having starting a startingpH of 7.0, 7.5 and 8.0 all led to good results when preparing nucleicacid templates according to the alkaline shock protocol, and thenamplifying and detecting those templates. These results supported usefulranges for the starting pH of a first composition that included thebiological sample undergoing specimen processing, the pH buffer, and thedetergent prior to addition of the alkaline composition. Notably,because this testing was performed using a co-amplifiable RNA internalcontrol, and because all tests yielded valid results, it was concludedthat RNA was stable over this range of starting pH conditions. Tosimplify the description of the invention, the Examples presented belowall employed a biological sample/buffer/detergent combination having astarting pH of about 7.5 prior to addition of an alkaline hydroxide.Again, success of the alkaline shock sample processing technique can beachieved over a wide starting pH range.

Treatment Methods—Alkaline and Detergent Conditions

In a preferred embodiment of the invented method, a first compositionthat includes a biological sample, a pH buffer and a detergent, iscombined with a second composition that includes an alkalinecomposition. The combination is mixed and preferably allowed to incubatewith an immobilized capture probe, and perhaps also a soluble captureprobe capable of forming a bridge between an immobilized probe and atarget nucleic acid of interest. As the method is typically practiced,alkaline hydroxide is added to a tube or other reaction vessel thatalready contains the first composition. In a highly preferredembodiment, the first composition either also includes, or is combinedwith the soluble capture probe and immobilized capture probe prior toaddition of the alkali hydroxide.

Substances which may be used as the alkaline composition to effect thealkaline shock may be any solid, liquid or gaseous agent which creates astrong alkaline solution when dissolved in aqueous solution. Strongbases are highly preferred alkaline compositions (hereafter referred togenerally as “alkaline hydroxides”) useful in connection with theinvention. Examples of preferred alkaline hydroxides that can be used tocarry out the invented sample preparation method include sodiumhydroxide, lithium hydroxide, potassium hydroxide, and the like.Although it is contemplated that solid alkaline compositions can becombined with the first composition that includes the buffered detergentand biological sample solution (as might be achieved by adding the firstcomposition to a tube already containing a measured amount of the dryalkaline hydroxide reagent), it is preferred to use alkalinecompositions in solution form.

To achieve the benefits of the invention, the amount of alkalinecomposition added to the composition that includes the biologicalsample, the pH buffer and detergent is critical, and can easily begauged by the final pH of the complete mixture. It is preferred that theamount of added alkaline hydroxide is sufficient to increase the pH ofthe resulting mixture by at least 0.2 pH units, but not so much as toraise the pH above 9.5, more preferably not above 9.2, more preferablynot above 9.0, still more preferably not above 8.8. Testing resultsconfirm that useful amounts of added alkaline hydroxide are thoseamounts that cause the final pH of the mixture to fall within aspecified range. The preferred final pH range is from between pH 8.0 and9.2, more preferably from between pH 8.2 and 9.2, and still morepreferably from between pH 8.2 and 8.8. As reiterated below, uniformlygood results were achieved when the final pH of the mixture followingthe alkaline shock fell in the range of from pH 8.2 to 9.2. This wastrue for both DNA and RNA templates. When the amount of added alkalinehydroxide caused the mixture to exceed a final pH of 9.5, poor resultswere achieved.

Preferred Detergents

Detergents that can be used in connection with the invention may beanionic detergents, non-ionic detergents, zwitterionic detergents, orcationic detergents. Of these, the anionic and non-ionic detergents arethe most preferred. The detergent concentration in the lysis/capturereagent is preferably between 0.01 and 15 wt. %, the particularlypreferred concentration ranging between 0.05 to 10 wt. %. Based on thedemonstrated use of 400 μl of lysis/capture reagent and 500 μl ofbiological sample, the final concentration of detergent in the mixedcomposition that includes buffer, detergent and biological sample,preferably falls in the range of from about 0.01 wt. % to about 6.7 wt.%. Strong anionic detergents, including sulfates of alkyl alcohols andN-acyl-amino acids are highly preferred. While the precise nature of thedetergent used for conducting the alkaline shock-based samplepreparation procedure is not believed critical, examples of particularlypreferred detergents include lithium lauryl sulfate (LLS), and sodiumdodecyl sulfate (SDS).

Treatment Period

In a preferred embodiment, an aliquot of an alkaline hydroxide solutionis combined in a reaction vessel with a composition that includes abiological sample, a buffer, a detergent, and, if sequence-specifictarget-capture is to be performed, one or more immobilizable captureoligonucleotides. After a period of from about one second to about onehour, the contents are agitated to ensure uniform mixing, and thetarget-capture process which involves the immobilization, whether director indirect, of a polynucleotide liberated from the biological sample,and an immobilized oligonucleotide follows. Of course, non-specifictarget capture also can be employed. To facilitate laboratoryproductivity, the length of time during which the target-capture step isperformed is desirably no longer than necessary. However, becausenucleic acids liberated from the biological sample will be stable in themixed composition subsequent to addition of the alkaline hydroxide,allowing the mixtures to stand for up to at least a few hours is notbelieved harmful to the target nucleic acids. Thus, the treatmentconditions associated with the alkaline shock are believed quite mild.

Plastic Containers Disposed in an Automated Analyzer

The invented method of sample preparation preferably is carried out in adisposable reaction vessel, such as a plastic tube, or a disposable unitcomprising a plurality of tubes held in a spaced-apart configuration.For example, the disposable reaction vessel is preferably positionedwithin an analytical device at the time that the alkaline hydroxidesolution is added, and the addition step is preferably carried out by amanual, or an automated or robotic pipetting device. In a highlypreferred embodiment, the disposable reaction vessel is loaded into theanalytical device, and a manual, or an automated or robotic pipettingdevice adds to the vessel an aliquot of the biological sample and analiquot of lysis/capture reagent which includes a pH buffer and adetergent for lysing or disrupting biological membranes, such as cellmembranes, viral envelopes, and the like. The lysis/capture reagentpreferably also contains an immobilizable capture oligonucleotide andinsoluble beads for capturing polynucleotides liberated from thebiological sample. Thereafter, the same or a different automated orrobotic pipetting device adds to the tube an aliquot of alkalinehydroxide solution. The contents of the tube can then be agitated toensure complete mixing, and the mixed sample incubated at a temperatureand for a period sufficient to permit capture of the liberatedpolynucleotides. Because the alkaline shock conditions are mild, thereis no substantial chemical degradation that is known to occur byextended or variable periods of standing, as may occur when differentanalytical protocols are executed on the automated analyzer in a singledaily cycle of laboratory testing.

Target Capture—Methods and Oligonucleotides

The disclosed alkaline shock-based sample preparation method has beendemonstrated to have particular value when coupled with a target captureprocedure that enriches the sample for nucleic acids. Separate preferredembodiments rely on non-specific target capture (i.e., where nucleicacids are captured in a manner substantially independent of the basesequence of the nucleic acids), and on sequence-specific target capture.Either or both of these methods can employ an immobilizable orimmobilized capture oligonucleotide.

Preferred capture oligonucleotides include a first sequence that iscomplementary to a polynucleotide containing a target sequence which isto be amplified, covalently attached to a second sequence (i.e., a“tail” sequence) that serves as a target for immobilization on a solidsupport. Any backbone to link the base sequence of a captureoligonucleotide may be used. In certain preferred embodiments thecapture oligonucleotide includes at least one methoxy linkage in thebackbone. The tail sequence, which is preferably at the 3′ end of acapture oligonucleotide, is used to hybridize to a complementary basesequence to provide a means for capturing the hybridized target nucleicacid in preference to other components in the biological sample.

Although any base sequence that hybridizes to a complementary basesequence may be used in the tail sequence, it is preferred that thehybridizing sequence span a length of about 5-50 nucleotide residues.Particularly preferred tail sequences are substantially homopolymeric,containing about 10 to about 40 nucleotide residues, or more preferablyabout 14 to about 30 residues. A capture oligonucleotide according tothe present invention may include a first sequence that specificallybinds a target polynucleotide, and a second sequence that specificallybinds an oligo(dT) stretch immobilized to a solid support.

Using the components illustrated in FIG. 1, one assay for detectingnucleic acid sequences in a biological sample includes the steps ofcapturing the target nucleic acid using the capture oligonucleotide,amplifying the captured target region using at least two amplificationoligonucleotides, or at least two primers, and detecting the amplifiednucleic acid by first hybridizing the labeled probe to a sequencecontained in the amplified nucleic acid and then detecting a signalresulting from the bound labeled probe.

The capturing step preferably uses a capture oligonucleotide where,under hybridizing conditions, one portion of the capture oligonucleotidespecifically hybridizes to a sequence in the target nucleic acid and atail portion serves as one component of a binding pair, such as a ligand(e.g., a biotin-avidin binding pair) that allows the target region to beseparated from other components of the sample. Preferably, the tailportion of the capture oligonucleotide is a sequence that hybridizes toa complementary sequence immobilized to a solid support particle.Preferably, first, the capture oligonucleotide and the target nucleicacid are in solution to take advantage of solution phase hybridizationkinetics. Hybridization produces a capture oligonucleotide:targetnucleic acid complex which can bind an immobilized probe throughhybridization of the tail portion of the capture oligonucleotide with acomplementary immobilized sequence. Thus, a complex comprising a targetnucleic acid, capture oligonucleotide and immobilized probe is formedunder hybridization conditions. Preferably, the immobilized probe is arepetitious sequence, and more preferably a homopolymeric sequence(e.g., poly-A, poly-T, poly-C or poly-G), which is complementary to thetail sequence and attached to a solid support. For example, if the tailportion of the capture oligonucleotide contains a poly-A sequence, thenthe immobilized probe would contain a poly-T sequence, although anycombination of complementary sequences may be used. The captureoligonucleotide may also contain “spacer” residues, which are one ormore bases located between the base sequence that hybridizes to thetarget and the base sequence of the tail that hybridizes to theimmobilized probe. Any solid support may be used for binding the targetnucleic acid:capture oligonucleotide complex. Useful supports may beeither matrices or particles free in solution (e.g., nitrocellulose,nylon, glass, polyacrylate, mixed polymers, polystyrene, silanepolypropylene and, preferably, magnetically attractable particles).Methods of attaching an immobilized probe to the solid support are wellknown. The support is preferably a particle which can be retrieved fromsolution using standard methods (e.g., centrifugation, magneticattraction of magnetic particles, and the like). Preferred supports areparamagnetic monodisperse particles (i.e., uniform in size±about 5%).

Retrieving the target nucleic acid:capture oligonucleotide:immobilizedprobe complex effectively concentrates the target nucleic acid (relativeto its concentration in the biological sample) and purifies the targetnucleic acid from amplification inhibitors which may be present in thebiological sample. The captured target nucleic acid may be washed one ormore times, further purifying the target, for example, by resuspendingthe particles with the attached target nucleic acid:captureoligonucleotide:immobilized probe complex in a washing solution and thenretrieving the particles with the attached complex from the washingsolution as described above. In a preferred embodiment, the capturingstep takes place by sequentially hybridizing the capture oligonucleotidewith the target nucleic acid and then adjusting the hybridizationconditions to allow hybridization of the tail portion of the captureoligonucleotide with an immobilized complementary sequence (e.g., asdescribed in PCT No. WO 98/50583). After the capturing step and anyoptional washing steps have been completed, the target nucleic acid canthen be amplified. To limit the number of handling steps, the targetnucleic acid optionally can be amplified without releasing it from thecapture oligonucleotide.

Useful capture oligonucleotides may contain mismatches to theabove-indicated sequences, as long as the mismatched sequences hybridizeto the nucleic acid containing the sequence that is to be amplified.

Useful Amplification Methods

Amplification methods useful in connection with the present inventioninclude: Transcription Mediated Amplification (TMA), Nucleic AcidSequence-Based Amplification (NASBA), the Polymerase Chain Reaction(PCR), Strand Displacement Amplification (SDA), and amplificationmethods using self-replicating polynucleotide molecules and replicationenzymes such as MDV-1 RNA and Q-beta enzyme. Methods for carrying outthese various amplification techniques respectively can be found in U.S.Pat. No. 5,399,491, published European patent application EP 0 525 882,U.S. Pat. No. 4,965,188, U.S. Pat. No. 5,455,166, U.S. Pat. No.5,472,840 and Lizardi et al., BioTechnology 6:1197 (1988). Thedisclosures of these documents which describe how to perform nucleicacid amplification reactions are hereby incorporated by reference.

In a preferred embodiment of the invention, target nucleic acidsequences are amplified using a TMA protocol. According to thisprotocol, the reverse transcriptase which provides the DNA polymeraseactivity also possesses an endogenous RNase H activity. One of theprimers used in this procedure contains a promoter sequence positionedupstream of a sequence that is complementary to one strand of a targetnucleic acid that is to be amplified. In the first step of theamplification, a promoter-primer hybridizes to the target RNA at adefined site. Reverse transcriptase creates a complementary DNA copy ofthe target RNA by extension from the 3′ end of the promoter-primer.Following interaction of an opposite strand primer with the newlysynthesized DNA strand, a second strand of DNA is synthesized from theend of the primer by reverse transcriptase, thereby creating adouble-stranded DNA molecule. RNA polymerase recognizes the promotersequence in this double-stranded DNA template and initiatestranscription. Each of the newly synthesized RNA amplicons re-enters theTMA process and serves as a template for a new round of replication,thereby leading to an exponential expansion of the RNA amplicon. Sinceeach of the DNA templates can make 100-1000 copies of RNA amplicon, thisexpansion can result in the production of 10 billion amplicons in lessthan one hour. The entire process is autocatalytic and is performed at aconstant temperature.

Kits

The invention also embraces kits that can be used for carrying outalkaline shock-based sample preparation procedures. Kits in accordancewith the invention will include in separate vials or containers: alysis/capture reagent and an alkaline hydroxide. In certain embodimentsof the invention, one or both of these reagents is a dry, lyophilized,or semi-solid composition which can be reconstituted with a liquidcomponent, such as water, prior to use. In certain highly preferredembodiments, the alkaline hydroxide composition requires reconstitutionwith a liquid agent prior to use. In other embodiments, the alkalinehydroxide is packaged in the kit as a liquid composition. Thelysis/capture reagent preferably includes a detergent and a buffer witha pH less than 8.0 upon reconstitution, if reconstitution is necessary.

Preferred Embodiments of the Invention

To ensure development of a general procedure for enhancing detectabilityof DNA targets without substantially compromising detectability of RNAtargets in nucleic acid amplification-based assays, certain aspects ofthe invention were created using a model multiplex assay essentially asdescribed in Example 7 of published International Patent Application No.PCT/US03/18993, the disclosure of which is incorporated by reference.This assay, which is capable of amplifying both RNA targets (HIV-1 andHCV) and a DNA target (HBV), employs a common set of primers foramplifying all of HBV subtypes A-C. Accordingly, the proceduresdescribed below essentially isolated sample preparation as a variable.

The model assay used in the procedures described herein involved threemain steps which all took place in a single tube: sample preparation;HIV-1 or HCV RNA or HBV DNA target amplification; and detection of theamplification products (amplicon) by the Hybridization Protection Assay(HPA). During sample preparation, viral RNA and DNA were isolated fromplasma specimens via the use of target capture. Plasma was combined witha buffered detergent solution to facilitate solubilization of the viralenvelope, denaturation of proteins and release viral genomic RNA and/orDNA from viral particles contained in the specimen. An alkaline shockeffected by the further addition of an alkaline hydroxide solutionserved as a test step during development of the invention. Forsimplicity, the buffered detergent solution that was combined with theplasma specimen was termed, “lysis/capture reagent.” Oligonucleotides(capture oligonucleotides) that were homologous to conserved regions ofHIV-1, HCV, and HBV were hybridized to the HIV-1 or HCV RNA or HBV DNAtargets, if present, in the test specimen. Hybridized targets were thencaptured onto magnetic microparticles, and separated from the bulkplasma in a magnetic field. Wash steps were used to remove extraneousplasma components from the reaction tube. Next, any captured viralnucleic acids were used as templates in a primer-dependent in vitronucleic acid amplification reaction. Target amplification in the modelassay occurred via TMA, a transcription-based nucleic acid amplificationmethod that uses two enzymes, MMLV reverse transcriptase and T7 RNApolymerase. The model assay was capable of amplifying regions of HIV-1RNA, HCV RNA, and/or HBV DNA. Detection of amplicons was achieved by HPAusing a mixture of single-stranded nucleic acid probes that werecomplementary to the amplicons. Each nucleic acid probe harbored achemiluminescent label, and hybridized specifically to one of theamplicons. A “selection” reagent differentiated between hybridized andunhybridized probes by inactivating the label on unhybridized probes.During the detection step, the chemiluminescent signal produced by thehybridized probe was measured in a luminometer and was reported asrelative light units (RLU).

The integrity of assay results was verified by the use of an InternalControl (IC) that was added to each test specimen, external control, orassay calibrator tube via the working lysis/capture reagent. The IC inthis reagent controlled for specimen processing, amplification, anddetection steps. The IC signal in each tube or assay reaction wasdiscriminated from the HIV-1/HCV/HBV signal by the differential kineticsof light emission from probes with different labels. The IC amplicon wasdetected using a probe with rapid emission of light (termed flashersignal). Amplicon specific for HIV-1/HCV/HBV was detected using probeswith relatively slower kinetics of light emission (termed glowersignal). Those having an ordinary level of skill in the art willappreciate that the Dual Kinetic Assay (DKA) is a standard method usedto differentiate between the signals from flasher and glower labels.When used for simultaneous detection of HIV-1, HCV, and HBV, the modelassay differentiated between IC and combined HIV-1/HCV/HBV signals, butdid not discriminate between individual HIV-1, HCV, and HBV signals.

The interpretation of assay results relied on signals representingdetection of one of the target nucleic acids as well as the IC. Morespecifically, two cutoffs were determined for each assay: one for theAnalyte signal (glower signal), termed the Analyte Cutoff, and one forthe IC signal (flasher signal), termed the IC Cutoff. For each sample,an Analyte signal RLU value and IC signal RLU value were determined.Analyte signal RLU divided by the Analyte Cutoff was termed the AnalyteSignal/Cutoff, or “S/CO.” For a sample with Analyte signal less than theAnalyte Cutoff (i.e., Analyte S/CO<1.00), the Internal Control (IC)signal must be greater than or equal to the Internal Control Cutoff (ICCutoff) for the result to be valid. In this case the Internal Controlresult will be considered to be valid, and the sample will be reportedas nonreactive. For a sample with the Analyte signal less than theAnalyte Cutoff (i.e., Analyte S/CO<1.00) and the Internal Control signalless than the Internal Control Cutoff, the Internal Control result willbe considered as invalid, and the sample result will be invalid. For allsamples, the Internal Control signal may not exceed 475,000 RLU. In suchan instance, the sample will automatically be reported as invalid.

As indicated above, the amplification technique used to illustrate theinvention was the Transcription Mediated Amplification. However, thedisclosed sample preparation method can be used in conjunction with anyin vitro nucleic acid amplification technique that will be familiar tothose having an ordinary level of skill in the art. This is because theinvented method operates to improve the lysis and target-capture stepswhich are independent of the nucleic acid amplification procedure.

The following Example describes preliminary experiments that empiricallyestablished the amount of an alkaline hydroxide solution that could beadded to a sample of lysis/capture reagent without completely exceedingthe buffer capacity of the mixture. In this instance, the exemplarybuffered detergent solution was a HEPES-buffered lithium lauryl sulfatesolution that included capture oligonucleotides and magnetic beads.These procedures were used to establish a pH profile only, and did notinvolve nucleic acid amplification. The HEPES(N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) component of thelysis/capture reagent has a pKa of about 7.5, and conventionally is usedfor buffering solutions in the range of from pH 6.8 to 8.2. Buffersother than HEPES can be used to carry out the invented samplepreparation procedure, provided that the added alkali solution is addedin an amount such that the final pH falls within the range specifiedbelow. Of course, it is preferred for the starting pH of the buffereddetergent solution to be approximately neutral to slightly alkaline,meaning in the range of from about pH 6.5 to about 8.0, or morepreferably from about pH 7.0 to about 8.0, or still more preferably fromabout pH 7.0 to about 7.5.

Example 1 describes the effect of adding an alkaline hydroxide solutionto a HEPES-buffered detergent solution that either included, or did notinclude an added plasma sample. The results from this procedure providedan empirical basis for determining the final pH of mixtures createdusing different amounts of the hydroxide solution.

Example 1 Determining the pH Effect of Adding Alkali to a BufferedDetergent Solution which Includes a Plasma Sample

Aliquots (400 μl) of a lysis/capture reagent (i.e., a buffered detergentsolution) were dispensed into plastic reaction tubes. The lysis/capturereagent contained soluble capture oligonucleotides and about 40 μg of0.7-1.05 t paramagnetic particles (Seradyn, Indianapolis, Ind.)covalently linked to poly-(dT₁₄). Capture oligonucleotides were capableof simultaneously hybridizing to the particle-bound poly-(dT) and to thenucleic acids of HBV subtypes-A, -B or -C. The lysis/capture reagentfurther included an HIV-1 internal amplification control template, HIV-1and HCV-specific capture oligonucleotides, about 800 mM HEPES (pH 7.5),and about 10% wt/vol lithium lauryl sulfate. Half of the reaction tubesalso received an aliquot (500 μl) of a processed control plasma that didnot contain fibrin. The tubes then received 100 μl aliquots of NaOHsolution having a concentration in the range of from 1.0 to 2.5N. Oneset of tubes containing lysis/capture reagent, or the combination oflysis/capture reagent and plasma sample were reserved as controls thatdid not receive an aliquot of NaOH solution. All samples were mixedusing a mechanical vortexer, and the pH values of the resulting mixtureswere determined using a Model 9100 pH meter from VWR Scientific Products(Chester, Pa.). Results from these procedures are presented in Table 1.

TABLE 1 Titration of Alkali into a Buffered Detergent Solution Combinedwith a Plasma Sample pH After Mixing Conc. of Sample: Sample: added NaOHLysis/Capture Reagent Lysis/Capture Reagent + Plasma None 7.45 7.45 1.0N 8.05 8.05 1.4 N 8.31 8.35 1.8 N 8.65 8.76 2.2 N 9.85 10.62 2.5 N 13.212.6

The results presented in Table 1 indicated that addition of 100 μl of anNaOH solution having a concentration of 2.2N or greater started toexceed the buffer capacity of the buffered detergent solution, with orwithout an added plasma sample. This was based on the observation thatthe final pH of the mixtures increased greater than one pH unit onlywhen concentrations of NaOH at 2.2N and higher were used. These resultsprovided a basis for additional studies aimed at quantifying the effectof added alkali on the ability to detect DNA targets in in vitroamplification reactions, and provided guidance useful for adapting theprocedure to the use of RNA targets which might be subject to hydrolysisunder conditions of elevated pH.

The following Example describes how varying amounts of added alkaliinfluenced detection of HBV subtype-B in an in vitro amplificationsystem that involved viral lysis, capture of released nucleic acids,amplification and detection steps. In this procedure, the monitoredassay parameters included % positivity for detection, % CV (coefficientof variability), and the mean quantitative signal strength (measured inrelative light units, or “RLU”). Those having an ordinary level of skillin the art will appreciate that low % CV values advantageously indicatehigher levels of assay precision, and so are greatly preferred.

Example 2 describes procedures that defined useful amounts of alkalithat could be added to a mixture of virus-containing plasma and alysis/capture reagent preliminary to conducting an in vitro nucleic acidamplification reaction.

Example 2 Sample Preparation that Includes Alkaline Shock Improves AssayPerformance

A 400 μl aliquot of the lysis/capture reagent described in Example 1 wascombined in a plastic reaction tube with 500 μl of a plasma sampleobtained from an individual infected with HBV subtype-B. Control tubesincluded virus-negative plasma instead of virus-positive samples. Allplasma samples used as the source of viral templates in this procedurehad been diluted 1:3 with virus-negative processed control plasma. Next,100 μl aliquots of NaOH solutions having different concentrations wereadded to the different tubes containing the combination of thelysis/capture reagent and plasma. The alkaline solutions used in theprocedure had NaOH concentrations ranging from 0.05 to 2.5N. A controltube included water in place of the NaOH solution. The mixtures werevortexed briefly to ensure mixing, heated to 60° C. for about 20minutes, and then cooled to room temperature for 15 minutes to allowhybridization and target capture. A magnetic field was applied tocollect the particle complexes containing the immobilized captureoligonucleotide and HBV DNA using procedures such as those described byWang in U.S. Pat. No. 4,895,650. The particles were then washed twicewith 1 ml of a washing buffer (10 mM HEPES at pH 7.5, 6.5 mM NaOH, 1 mMEDTA, 0.3% (v/v) ethanol, 0.02% (w/v) methyl-paraben, 0.01% (w/v)propyl-paraben, 150 mM NaCl, 0.1% (w/v) sodium lauryl sulfate). Washedparticles were resuspended in 75 μl of an amplification reagent, and thecontents of the tube overlaid with inert oil to prevent evaporation. Theamplification reagent included salts, nucleotides, ribonucleotides,HBV-specific primers, as well as primers capable of amplifying HIV-1 andHCV target sequences. After vortexing briefly, the mixture was firstincubated at 60° C. for 10 minutes to facilitate primer annealing, andthen equilibrated at 41.5° C. for 10 minutes. Aliquots of pre-warmedenzyme reagent that included Moloney Murine Leukemia Virus (MMLV)reverse transcriptase (5,600 units/reaction) and T7 RNA polymerase(3,500 units/reaction) were then added to the mixtures. After a one hourincubation at 41.5° C., the reaction was complete and HBV amplificationproducts were detected using an acridinium ester-labeled hybridizationprobe in a homogenous protection assay, essentially as described underExample 1 of published International Patent Application No.PCT/US03/18993. Reactions that gave positive signals when hybridizedwith a probe specific for the internal control amplicon, or with a probespecific for the HBV amplicon, were scored as valid runs. In order for avalid run to be considered positive for the presence of HBV amplicons,the chemiluminescent signal indicating probe hybridization must haveexceeded 50,000 RLU in an assay. Results from these procedures arepresented in Table 2, and in FIGS. 2A-2C.

TABLE 2 Optimizing Conditions for Alkaline Shock Plasma Additive Sample% Positive CV % Mean RLU Water control 0 37 2231 (control) HBV-B 1:3 10144 296066 0.05N NaOH  control 0 23 2709 HBV-B 1:3 10 195 232295 0.1NNaOH control 0 33 2248 HBV-B 1:3 50 64 721476 0.3N NaOH control 0 203546 HBV-B 1:3 60 34 898200 0.6N NaOH control 0 37 4023 HBV-B 1:3 60 36930407 1.0N NaOH control 0 17 5017 HBV-B 1:3 90 10 1136773 1.4N NaOHcontrol 0 42 4387 HBV-B 1:3 100 4 1183610 1.8N NaOH control 0 24 4216HBV-B 1:3 100 5 1184132 2.2N NaOH control 0 16 4559 HBV-B 1:3 90 81166869 2.5N NaOH control 0 211 13087 HBV-B 1:3 0 47 5931

The results from these procedures indicated that sample preparationwhich included an alkaline shock step advantageously led to dramaticallyimproved assay performance. Notably, the fact that all of the assaysdiffered only by the nature of the alkaline shock step confirmed thatthe benefits achievable by the use of an alkaline shock did not dependon the particular oligonucleotides used in the procedure. Indeed, thecontrol trial that received an aliquot of water instead of NaOH solutiongave relatively low % positivity levels and undesirably high % CVvalues. Conversely, an alkaline shock performed using an NaOH solutionhaving a concentration in the range of from 0.1 to 2.2N gavedramatically greater % positivity levels with increased precision, asjudged by the reduced % CV values.

Importantly, these results also provided evidence for an optimal rangeof hydroxide concentrations that could be used in the alkaline shockprocedure. More specifically, the data revealed that use of the highestconcentration of NaOH substantially compromised assay performance to thepoint where % positivity was reduced to zero. This amount of hydroxidesolution, when mixed with the lysis/capture reagent and plasma sample,yielded a final pH of 12.6 (see Table 1). Thus, adding an amount ofalkaline hydroxide sufficient to result in a final pH of 12.6 eliminatedthe ability of the assay to detect the target. Conversely, addition ofan NaOH solution in an amount sufficient to raise the final pH to arange of from about 8.0 to about 10.6 gave good results. In thisexperiment, the best results were achieved by adding NaOH solution in anamount sufficient to raise the final pH to a range of from about 8.3 toabout 8.8. Notably, this range was nearly identical to the preferredrange of from pH 8.2 to 9.2 established in Example 8, below. Theseranges define preferred pH ranges that result from the addition ofappropriate amounts of an alkaline solution, preferably an alkalinehydroxide solution, to a buffered detergent solution containing abiological sample.

The following procedures proved that a transient high pH (i.e., an“alkaline shock”) was required to achieve improved assay sensitivity. Inthis Example, the order in which three reagents were combined was variedto investigate whether the beneficial effects described herein resultedfrom changing the final pH of the sample, or from a different mechanism.

Example 3 describes procedures which proved the beneficial effects ofthe alkaline shock method derive from a transient exposure to alkalineconditions.

Example 3 A Transient Alkaline Shock is Required to Improve AssayPerformance

Sample preparation methods that involved combining three reagents (analkaline hydroxide, a plasma sample, and a lysis/capture reagent) indifferent orders were used to address the mechanism of action underlyingthe observed assay improvement. The reagents and their amounts used inthe procedures were: 400 μl of lysis/capture reagent, 500 μl of a plasmasample containing HBV subtype-B virus particles (i.e., virus-infectedplasma from a human donor diluted 1:10 with virus-negative processedplasma), and 100 μl of 1.6N NaOH. The concentration of the alkalinehydroxide solution was selected because it was within the range of thoseyielding good results in the preceding Example. All reagents werepipetted into plastic reaction tubes, and target-capture, amplificationand detection were carried out as described in the preceding Examples. Acontrol reaction conducted using 1:10 diluted HBV-positive plasma andlysis/capture reagent without any added NaOH gave 40% positivereactivity, thereby defining a standard for comparison. Negative controlreactions conducted using HBV-negative plasma in place of theHBV-positive serum samples (“HBV Sample” in Table 2) for all of theconditions listed in the table uniformly gave 0% positive reactivity inthe amplification and detection reaction, as expected. Procedurescarried out using 1:20 diluted HBV-positive plasma samples in place ofthe 1:10 diluted samples gave results consistent with those presented inTable 3. The order of addition of the three reagents for each trialcondition (n=10) is given in the table.

TABLE 3 A Transient Alkaline Shock Produces Beneficial Results % FirstReagent Second Reagent Third Reagent Positive Lysis/Capture HBV SampleAlkali 100 Reagent Alkali HBV Sample 20 HBV Sample Lysis/Capture ReagentAlkali 90 Alkali Lysis/Capture Reagent 100 Alkali Lysis/Capture ReagentHBV Sample 40 HBV Sample Lysis/Capture Reagent 80

The results in Table 3 indicated that the order of reagent additionprofoundly influenced the assay outcome. First combining thelysis/capture reagent and alkaline hydroxide solution with each other,regardless of the order of addition of these reagents to the reactiontube, gave results similar to the control that omitted the alkalineshock. Thus, adding the virus-containing sample after the lysis/capturereagent and alkaline hydroxide were already combined yielded no benefitmeasurable by % positivity. Conversely, excellent results were achievedby first combining the lysis/capture reagent and the sample containingHBV virus particles (in either order), and thereafter combining thealkaline hydroxide solution with that mixture. This highly preferredorder of addition advantageously avoided direct exposure of thebiological sample to the concentrated hydroxide solution, and soadvantageously should minimize alkaline hydrolysis of RNA templates.

While not wishing to be bound by any particular theory of operation, theforegoing results support a mechanism wherein a local, transient high pHwithin the reaction tube containing the lysis/capture reagent and virussample had some effect which resulted in the advantages disclosedherein. Because success of the technique result from transient high pHexposure, the method disclosed herein has been termed, “alkaline shock.”

The results presented above also indicated that the final pH of thereaction mixtures (as described in Example 1) can be used to gauge theamount of alkaline solution needed to produce good results, but that thefinal pH of the mixture did not predict success of the procedure.Indeed, if the final pH of the mixture determined the outcome of theassay, then all of the trial conditions presented in Table 3 would haveyielded identical results, and that was not the case. Accordingly,preferred modes of carrying out the invention involve adding an alkalinesolution to a sample that includes a pH buffer, a detergent and abiological sample to be tested for the presence of a particular nucleicacid. In a highly preferred embodiment, the biological sample is a bodyfluid, such as whole blood, plasma, serum, and the like.

The following Example employed a statistical analysis to measure howassay sensitivity for different HBV subtypes was improved by includingan alkaline shock during the sample preparation procedure. For thepurpose of this demonstration, a multiplex assay essentially asdisclosed under Example 7 of published International Patent ApplicationPCT/US03/18993, was employed with the only substantive difference beingthe addition of an alkaline shock step during the sample preparationprocedure.

Example 4 describes how the alkaline shock technique improvedquantitative assay performance for multiple HBV subtypes.

Example 4 Quantifying Effects of the Alkaline Shock Technique

Panels of plasma samples containing known quantities of HBV subtype-A,-B or -C viral particles were produced by methods that will be familiarto those having an ordinary level of skill in the art. Samples wereprepared using an alkaline shock protocol in which 400 μl oflysis/capture reagent and 500 μl of individual panel members were firstcombined in plastic reaction tubes, 100 μl of 1.6N NaOH was added, andthe tubes agitated thereafter to ensure complete mixing. Target capture,amplification and detection of amplification products were carried outas described above. Control reactions that omitted the alkaline shockprocedure were performed in parallel. Regression analysis using theProbit function in SAS® System software (version 8.02) (Cary, N.C.) wasused to calculate the 95% and 50% detection levels. Invalid reactionswere not re-tested and were not included in the analysis of analyticalsensitivity. Results from these procedures are presented in FIGS. 3A-3C,and the Probit Analysis is summarized in Table 4.

TABLE 4 Quantifying the Effects of Alkaline Shock on HBV SubtypeDetection HBV Assay Sensitivity Fold Geno- Detection (copies/ml)Increase in type Probability Control Alkaline Shock Sensitivity B 95% 1405 (1168-1792)  78 (61-116)  18x 50% 504 (440-623) 22 (14-29)  23x C95% 98 (78-134) 26 (21-35) 3.8x 50% 37 (29-49)  10 (8-13)  3.7x A 95% 74(58-108) 40 (32-57) 1.9x 50% 26 (20-34)  12 (9-16)  2.2x 95% confidenceintervals are shown in parentheses

The results summarized in Table 4 confirmed that the alkaline shockprocedure enhanced detectability of all three subtypes of HBV, althoughto somewhat different extents. Using conventional procedures that didnot employ an alkaline shock, assay sensitivity for the subtype-B virusat 95% detection probability was lower than the other subtypes by about16 fold. Using the improved sample preparation method that incorporatedan alkaline shock procedure prior to target capture and amplificationdramatically improved assay performance to the point where all of thesubtypes could be detected at levels below 100 copies/ml of sample.Interestingly, the alkaline shock sample preparation method improvedassay sensitivity for the subtype-B virus most dramatically. Thisdifferential improvement could not have been predicted in advance ofthis showing, and may provide insight into the mechanism underlying theeffect of the invented method.

Results presented in the preceding Examples showed that a transientalkaline shock during the sample preparation procedure dramaticallyimproved subsequent detection of a nucleic acid target. Testingdescribed in the following Example addressed the mechanism underlyingthis improvement. More specifically, an experiment was carried out toinvestigate whether addition of the alkaline hydroxide solution to themixture of lysis/capture reagent and biological sample had only adenaturing effect on proteins and nucleic acids in the sample, therebyincreasing availability of the viral nucleic acids for subsequentamplification and detection. Although alkaline conditions are known todenature proteins and nucleic acids, the results presented belowindicated that the beneficial results observed by the proceduresdescribed herein were not fully explained by alkaline denaturation.

Example 5 describes procedures which proved that the alkaline shockphenomenon was not primarily mediated by alkaline denaturation ofproteins and/or nucleic acids.

Example 5 Alkaline Shock Requires the Combined Effects of Alkali andDetergent

Specialized lysis/capture reagents were prepared to contain either 0%,5% or 10% lithium lauryl sulfate (LLS) detergent. Notably, all otherinstances of lysis/capture reagent described herein were prepared using10% LLS. Aliquots (400 μl) of one of the target-capture reagents werefirst combined with aliquots (500 μl) of a 1:10 dilution of a serumsample obtained from a patient infected with HBV subtype-B. All trialsincluded approximately 100 copies of the HBV genome. Thereafter, trialsthat were to be treated with alkaline hydroxide received 100 μl of 1.6 NNaOH, and were vortexed briefly. Control trials received 100 μl of waterinstead of NaOH solution. Target capture, amplification and detection ofamplification products were carried out as described above. All trialswere carried out in replicates of 10. Results from these procedures arepresented in Table 5.

TABLE 5 Alkaline Shock Requires the Combination of a Detergent andAlkali % Detergent in Lysis/Capture % Detection of HBV Reagent ControlAlkaline Treatment 0 0 0 5 10 80 10 20 90

The results presented in Table 5 indicated that the combination ofdetergent and alkali were required to effect the alkaline shock. Indeed,samples prepared using lysis/capture reagent that omitted detergentfailed to detect the HBV analyte, even in trials that received thealkaline hydroxide. Thus, treatment with alkali in the absence ofdetergent under the specified pH conditions was not sufficient to yieldgood results. This indicated that the mechanism by which alkaline shockwas effected was not entirely due to alkaline-mediated denaturation ofproteins and nucleic acids. Instead, there was a synergistic effect thatrequired first combining the biological sample with buffer anddetergent, and thereafter adding the alkaline hydroxide.

In addition to procedures carried out using HBV particles as a modelsource of DNA targets, additional experiments were performed toinvestigate the effect of an alkaline shock during sample preparation onRNA targets. As in the preceding Example, a multiplex assay capable ofamplifying and detecting HIV-1, HCV, and HBV nucleic acids was used toexamine the effect of alkaline shock on RNA targets. Indeed, the knownsensitivity of RNA to hydrolysis suggested that the alkaline shockprocedure would only be useful in connection with the isolation of DNAtargets preliminary to amplification and detection.

Example 6 describes procedures which demonstrated that RNA targets couldbe amplified and detected using nucleic acid templates prepared inprocedures that included an alkaline shock.

Example 6 Effect of Alkaline Shock on Detection of HIV-1 and HCV

Panels of plasma samples having known quantities of one of the followingRNA viruses were produced by standard methods: HCV-1a, HCV-2b, andHIV-1b. Samples were prepared using an alkaline shock protocol in which400 μl of lysis/capture reagent and 500 μl of individual panel memberswere first combined in plastic reaction tubes, 100 μl of 1.6N NaOH wasadded, and the tubes agitated thereafter to ensure complete mixing.Target capture, amplification and detection of amplification productswere carried out essentially as described above, using appropriatedetection probes at the conclusion of the amplification procedure.Control reactions that omitted the alkaline shock procedure wereperformed in parallel. Except for trials conducted using the highestviral titers, all reactions were performed using replicates of 20.Reactions conducted using the equivalent of 300 copies/ml of plasmasample were performed using replicates of 10. Results from theseprocedures are summarized in Tables 6-8, and in FIGS. 4A-4C. Table 9presents results from a Probit Analysis of the data in Tables 6-8.

TABLE 6 Effect of Alkaline Shock on HCV-1a Amplification and DetectionHCV-1a Sample % Number Mean IC Mean Amplicon No. (RNA copies/ml)Positive of Trials RLU RLU Invalid Control 300 100 10 185354 1453346 0100 100 20 182303 1368094 0 30 95 20 185996 1165246 0 10 52.6 20 199793458035 1 3 38.9 20 201781 352694 2 Alkaline 300 100 10 162777 1398738 0Shock 100 100 20 170729 1411556 0 30 95 20 170932 1157907 0 10 75 20179854 865606 0 3 46.7 20 182010 530033 5

TABLE 7 Effect of Alkaline Shock on HCV-2b Amplification and DetectionHCV-2b Sample % Number Mean IC Mean Amplicon No. (RNA copies/ml)Positive of Trials RLU RLU Invalid Control 300 100 10 265152 1272494 0100 100 20 251077 1190681 0 30 95 20 241570 911405 0 10 73.7 20 236737514461 0 3 15 20 235306 67760 1 Alkaline 300 100 10 242148 1287926 0Shock 100 100 20 260091 1020347 0 30 90 20 246163 530746 0 10 55 20232982 244802 0 3 5.3 20 224816 43514 1

TABLE 8 Effect of Alkaline Shock on HIV-1b Amplification and DetectionHIV-1b Sample % Number Mean IC Mean Amplicon No. (RNA copies/ml)Positive of Trials RLU RLU Invalid Control 300 100 10 11883 35509 0 100100 20 11824 103981 0 30 100 20 22536 179092 1 10 80 20 7814 115883 0 340 20 4167 52622 0 Alkaline 300 100 10 11738 19499 0 Shock 100 100 2013823 110696 0 30 90 20 10468 166067 0 10 68 20 22082 169677 1 3 21 2021784 53292 1

The results presented in Tables 6-8, and in FIGS. 4A-4C indicated thatany effect of the alkaline shock treatment of samples being tested forthe presence of RNA viruses was very minor. For instance, assaysensitivity for HCV-1a appeared to increase slightly, while thesensitivity for HCV-2b and HIV-1b may have decreased slightly. Notably,it is unclear whether these differences, which were noted only at verylow viral titers, were statistically significant. Overall, the resultsconfirmed that an alkaline shock could be integrated into a singlesample preparation procedure for isolating RNA and DNA targets. It wassomewhat surprising that the treatment could be gentle enough to permitsubsequent detection of RNA targets while being adequate to providesubstantial enhancement of DNA targets.

TABLE 9 Quantifying the Effects of Alkaline Shock on Detection of RNATargets RNA Detection Assay Sensitivity (copies/ml) Taret ProbabilityControl Alkaline Shock HCV-1a 95% 32 (22-69) 28 (18-93) 50% 8 (0-13) 3(0-8) HCV-2b 95% 23 (16-52) 31 (23-54) 50% 8 (4-13) 13 (8-19)  HIV-1b95% 16 (11-42) 32 (23-65) 50% 5 (0-8)  9 (3-15) 95% confidence intervalsare shown in parentheses

The tabulated results from the Probit Analysis, presented in Table 9,indicated that the alkaline shock treatment did not substantially impairdetectability of the RNA targets. Thus, the same alkaline shockconditions that enhanced detection of the HBV subtype-B nucleic acid didnot substantially compromise detection of RNA targets.

The foregoing Example demonstrated that sample preparation procedureswhich incorporated an alkaline shock could be used to isolate RNAtemplates, in addition to DNA templates. This result was somewhatsurprising because RNA is known to be subject to hydrolysis underalkaline conditions. The following Example confirmed this susceptibilitywhen the sample preparation procedure was varied such that a biologicalsample containing a known amount of HIV-O virions was first combinedwith an alkaline hydroxide prior to addition of a pH buffer anddetergent.

Example 7 describes procedures that were followed to assess the effectof alkaline treatment on the integrity of an RNA target. Results fromthese procedures showed that RNA hydrolysis was very rapid followingcontact between the biological sample containing HIV-O virions and thealkaline hydroxide solution.

Example 7 Order of Reagent Addition Profoundly Affects RNA Integrity

Serum samples containing HIV-O virions were diluted to known titersusing virus-negative serum. Aliquots (500 μl) of virion-containing serum(i.e., containing 30 copies/ml) were first deposited into plasticreaction tubes. Thereafter, 100 μl aliquots of 1.8 N LiOH were added andthe tubes allowed to stand for variable periods of time. Next, analiquot (400 μl) of lysis/capture reagent was added and vortexedbriefly. The delay time between addition of the alkaline hydroxidesolution and the pH buffered detergent solution (i.e., the lysis/capturereagent) ranged from 0 minutes to 1 hour. Surviving RNA templates werecaptured, amplified, and detected essentially as described above.Notably, the pH-buffered lysis/capture reagent in this procedureincluded a synthetic HIV-1 transcript that served as the internalamplification control. All trials were conducted in replicates of 10.

TABLE 10 Assessing the Effect of Alkali on RNA Integrity Time Delay % CV# # % Sample (mins) IC RLU Trials Invalid Positive Neg. Serum 0 13 10 00 HIV-O 0 5 10 0 10 30 copies/ml 2.5 9 10 0 0 5 3 10 0 0

The results presented in Table 10 indicated that hydrolysis of the HIV-ORNA target was very rapid when the alkaline hydroxide solution was addedto the biological sample in the absence of a pH buffer. Notably, resultsfrom preliminary testing indicated that the HIV-O target was detected at100% efficiency at the 30 copy/ml level when the alkaline hydroxide waseither omitted from the procedure or added after the virus-containingsample was first combined with the lysis/capture reagent (i.e., a pHbuffered detergent solution). As indicated in the table, only one of tenreplicates yielded detectable RNA templates when virus-containing serumwas mixed with the alkaline hydroxide solution and immediatelythereafter neutralized by the addition of lysis/capture reagent whichincluded a pH buffer. Other time points which extended the delay betweenaddition of the pH buffered solution to the alkali-treated virus samplesby up to one hour also yielded no detectable RNA templates, and so havebeen omitted from the table. The fact that the RNA internal controlsurvived the procedure, as judged by the fact that all of the trialswere considered valid, was expected. Taken together, these resultsconfirmed that mixing a biological sample containing an RNA target withan alkaline hydroxide solution prior to addition of a pH buffercompletely compromised the integrity of the RNA template. On the otherhand, first combining the RNA template with a pH buffer prior to mixingwith the alkaline hydroxide solution preserved template integrity.

Given the showing that RNA and DNA targets could be detected inmultiplex assays using a shared sample preparation method thatincorporated an alkaline shock, it was of interest to explore more fullythe effect of pH during the sample preparation procedure on final assayperformance. As indicated below, when the amount of alkaline hydroxideused in the sample preparation procedure resulted in a final pH greaterthan 9.5, the assay performed poorly. Notably, the procedures describedin the following Example employed LiOH in place of NaOH to effect thealkaline shock, and so also illustrated that the identity of thealkaline hydroxide used in the procedure was not critical for success ofthe sample preparation method.

Example 8 describes procedures that were followed to determine the upperlimit of the useful pH range for performing alkaline shock-based samplepreparation procedures.

Example 8 Optimizing the Amount of Alkaline Hydroxide Used forConducting the Alkaline Shock

Biological samples containing RNA or DNA viral targets were tested inthe model multiplex assay to determine how assay performance wasinfluenced by the final pH of a mixture that included a virus-containingplasma or serum sample, a buffered detergent solution (i.e., alysis/capture reagent), and an alkaline hydroxide during the samplepreparation procedure. In this procedure, 1.9N LiOH was used in place of1.6N NaOH to effect the alkaline shock. Preliminary procedures todetermine the final pH that resulted from the addition of differentamounts of the LiOH solution were conducted in replicates of three, andthe pH averages determined from those readings. In these preliminaryprocedures, 400 μl of lysis/capture reagent was combined with 500 μl ofvirus-negative serum and different volumes of 1.9N LiOH, and the finalpH of the mixtures determined as described above. The biological samplestested were: (1) HIV-1 O group positive plasma containing the equivalentof 30 copies/ml of the viral RNA; (2) HCV-1a positive plasma containingthe equivalent of 30 copies/ml of the viral RNA; and (3) HBV subtype Bdiluted into virus-negative control serum to a level of less than 200copies/ml. Virus-negative serum served as a control in the procedure. Inall instances, the biological sample was first combined with thelysis/capture reagent, and an aliquot of the alkaline hydroxide solutionadded and mixed thereafter. The number of valid and invalid runs wasscored following the amplification and detection procedure, and thepositively reacting trials determined as a percentage of the valid runs.All reactions were conducted using replicates of ten. A separateprocedure involved essentially similar methods, but focused on theaddition of LiOH in amounts that resulted in a slightly different pHrange during the sample preparation step, and tested HBV subtypeB-containing serum samples, HBV subtype C-containing serum samples, andHIV-1 O group positive plasma. The results from these procedures aresummarized in Tables 11 and 12.

TABLE 11 Establishing the Upper Limit of a Useful pH Range Vol. 1.9N %No. No. No. Avg. pH Sample LiOH (μl) pos Invalid Valid Reactive (n = 3)HIV-1 O 100 100 0 10 10 8.6 group HCV-1a 100 0 10 10 HBV-B 100 0 10 10Neg. Serum 0 0 10 0 HIV-1 O 120 100 2 8 8 9.2 group HCV-1a 100 3 7 7HBV-B 100 2 8 8 Neg. Serum 0 0 10 0 HIV-1 O 122.5 75 2 8 6 9.5 groupHCV-1a 88 1 9 8 HBV-B 100 2 8 8 Neg. Serum 0 5 5 0 HIV-1 O 125 0 8 1 010.7 group HCV-1a 0 10 0 0 HBV-B 100 7 3 3 Neg. Serum 0 8 2 0

TABLE 12 Establishing the Upper Limit of a Useful pH Range Vol. 1.9NAvg. pH Sample LiOH (μl) % pos No. Invalid No. Valid (n = 3) HBV-B 70100 0 10 8.02 85 100 0 10 8.22 100 90 0 10 8.46 115 100 0 10 8.73 130none 10 0 10.08 HBV-C 70 60 0 10 8.02 85 70 0 10 8.22 100 100 0 10 8.46115 100 0 10 8.73 130 none 10 0 10.08 HIV-1 O 70 80 0 10 8.02 group 8590 0 10 8.22 100 70 0 10 8.46 115 80 0 10 8.73 130 none 10 0 10.08

The results presented in Table 11 confirmed that HBV could be detectedat all pH levels tested, and established the upper limit of a useful pHrange for assays that included an RNA analyte or RNA internal control.More specifically, Table 11 shows that reactions conducted using HBVsubtype-B as the target gave 100% detectability in valid reactions atall levels of added alkaline hydroxide. However, the number of reactivetrials dropped below one-half under the highest pH condition for the HBVanalyte. Thus, it is less preferred to conduct alkaline shock-basedsample preparation when the amount of added alkaline hydroxide causesthe pH to exceed pH 10, and when the method includes a step forcapturing analyte nucleic acids preliminary to amplification. Assaysdesigned to detect RNA targets, or that rely on controls or calibratorsthat include RNA showed a distinctly different sensitivity to the pHconditions when compared with the results using the DNA target. When theamount of alkaline hydroxide used to effect the alkaline shock resultedin a mixture having a final pH of 9.5 or greater, the number of invalidruns increased dramatically when compared with the results of trialsperformed using final pH conditions of 9.2 and lower. Moreover, the %positive results indicated that trials conducted using the HIV-1 O groupand HCV-1a RNA targets were detected in 100% of reactions yielding validresults when the final pH of the mixture was 9.2 or lower. The %positive values dropped somewhat when the final pH was 9.5, and werefully compromised when the final pH was 10.7. While not wishing to bebound by any particular theory of operation, these results wereconsistent with a decrease in the efficiency of capturing intact targetsfor subsequent amplification when the final pH exceeded about pH 9.5.

The results presented in Table 12 indicated that all runs were valid,and that good results were achieved when the amount of alkalinehydroxide added to a mixture of biological sample and buffered detergentsolution (i.e., lysis/capture reagent) was sufficient to result in a pHfalling in the range of from about pH 8.0 up to less than about pH 10.Excellent results were achieved when the amount of added alkalinehydroxide was sufficient to result in a pH falling in the range of fromabout pH 8.0 up to about pH 8.7. The procedures that yielded the resultsappearing in Table 12 did not clearly establish the upper limit ofalkaline hydroxide that could be used to effect the alkaline shock. Thatdetermination was made based on the results in Table 11.

Based on the aggregated results presented herein, there was a preferredupper limit to the pH of a mixture that included a buffered detergentsolution (i.e., a lysis/capture reagent), a biological sample (such as asample of blood, plasma or serum), and an alkaline hydroxide. Moreparticularly, the amount of alkaline hydroxide used in the mixturepreferably yields a final pH of at least pH 8.0, but should not exceedabout pH 10.0. More preferably, and especially when RNA targets are tobe captured and amplified, the amount of alkaline hydroxide used in themixture preferably should yield a final pH of at least pH 8.0 but lessthan pH 9.5. Still more preferably, the amount of alkaline hydroxideused in the mixture preferably should yield a final pH of at least pH8.0, but should be equal to or less than about pH 9.2 (i.e., a final pHin the range of pH 8.0-9.2). Still more preferably, the amount ofalkaline hydroxide used in the mixture preferably should yield a finalpH of at least pH 8.0 but equal to or less than about pH 8.73 (i.e., afinal pH in the range of pH 8.0-8.73). Yet still more preferably, theamount of alkaline hydroxide used in the mixture preferably should yielda final pH of at least pH 8.0 but equal to or less than about pH 8.6(i.e., a final pH in the range of pH 8.0-8.6). In all instances, it waspreferred for the combination of the biological sample, the buffer, andthe detergent (i.e., prior to addition of the alkaline composition usedto effect the alkaline shock) to have a pH falling in the range of from6.5 to 8.0, more preferably in the range of from pH 7.0 to 8.0, morepreferably in the range of from 7.0 to about 7.5. Indeed, when thecombination of the biological sample, the buffer, and the detergentyielded a pH falling in the range of from pH 6.5-8.0, when the additionof the alkaline composition to effect the alkaline shock produced anincrease of atleast 0.2 pH units, and when the final pH of the mixturefollowing the alkaline shock fell in the range of from pH 8.2 to 9.2,excellent results were achieved for preparation of both DNA and RNAtemplates.

The foregoing demonstrations focused on the benefits of using thealkaline shock technique during the preparation of nucleic acids fromRNA and DNA viruses. The following Example describes how the samealkaline shock technique has been extended to the preparation of nucleicacids from bacteria. In this illustration, nucleic acids were preparedfrom Streptococcus agalactiae, a member of the Group B Streptococci(GBS). Those having an ordinary level of skill in the art willappreciate that GBS bacteria are known to be difficult to lyse. Thus,the illustration presented below represents a stringent test of thealkaline shock sample preparation method, and can be taken as indicatingthat the technique is useful for preparing nucleic acids from anybacterial species.

Example 9 details procedures that were used to prepare and detectnucleic acids from bacteria. The procedure included a fully integratedlysis, target capture, amplification and detection protocol conductedwith and without the alkaline shock step. This isolated the effect ofthe alkaline shock procedure with respect to the preparation of nucleicacid templates from bacteria. In vitro amplification was carried outusing a Transcription Mediated Amplification (TMA) protocol.

Example 9 Preparation and Detection of Bacterial Nucleic Acids

Cultured S. agalactiae bacteria were employed for testing the efficiencyof sample preparation using the alkaline shock technique. Bacteria grownovernight in a liquid culture medium were collected by gentlecentrifugation, and then washed 10 times in PBS to remove residualtraces of nucleic acids that may have been released into the culturemedium. The resulting bacterial pellet was taken up in PBS and thenserially diluted. Aliquots of the different dilutions were spread ontoblood agar plates to determine accurate titers. Remaining portions ofthe samples were used for preparing nucleic acids with and withoutalkaline shock, and the prepared samples used as sources of nucleic acidtemplates in TMA amplification reactions. Aliquots (400 μl) of alysis/capture reagent containing 4 pmols of a capture oligonucleotidehaving SEQ ID NO:1, together with about 40 μg 0.7-1.05 t paramagneticparticles (Seradyn, Indianapolis, Ind.) covalently linked to poly-(dT₁₄)were combined with aliquots (500 μl) of diluted GBS containing knownnumbers of organisms in plastic reaction tubes. The captureoligonucleotides were capable of simultaneously hybridizing to theparticle-bound poly-(dT) and to the bacterial rRNA. Samples wereprepared in replicates of nine for each level of bacteria undergoingtesting. Control tubes included with each set included 500 μl of PBScontaining 1,000 copies of purified bacterial rRNA instead of bacterialcells. Each tube then received 100 μl of either a water control, or 1.6N NaOH to effect the alkaline shock. After vortexing for 10 seconds, themixtures were incubated at 60° C. in a water bath for 15 minutes,followed by incubation at room temperature for another 15 minutes toallow hybridization and target capture onto the magnetic particles. Asdescribed above, a magnetic field was applied to collect the particlecomplexes containing the immobilized capture oligonucleotide and rRNA,and the collected particles washed twice with 1 ml of a washing buffer.Washed particles were resuspended in 75 μl of an amplification reagent,and the contents of the tube overlaid with inert oil to preventevaporation. As above, the amplification reagent included salts,nucleotides, ribonucleotides and about 5 μmol/reaction each of tworRNA-specific primers having the sequences of SEQ ID NO:3 and SEQ IDNO:2. After vortexing briefly, the mixture was incubated at 60° C. for10 minutes to facilitate primer annealing, and then equilibrated at41.5° C. for 10 minutes. Aliquots of pre-warmed enzyme reagent thatincluded Moloney Murine Leukemia Virus (MMLV) reverse transcriptase(5,600 units/reaction) and T7 RNA polymerase (3,500 units/reaction) werethen added to the mixtures. After a one hour incubation at 41.5° C., thereaction was complete and rRNA amplification products were detected in astandard homogenous protection assay, essentially as described underExample 1 of published International Patent Application No.PCT/US03/18993 using an acridinium ester-labeled hybridization probehaving the sequence of SEQ ID NO:4. The sequences of the relevantoligonucleotides employed for amplifying and detecting GBS bacteriaappear in Table 13.

TABLE 13 Oligonucleotides for Detecting a Bacterial Target Nucleic AcidOligo Oligo Function Oligo Sequence Identifier TargetGUUACGGGGCCAUUUUGCCGAGUUCCTTT SEQ ID NO:1 CaptureAAAAAAAAAAAAAAAAAAAAAAAAAAAAA A T7 AATTTAATACGACTCACTATAGGGAGAGA SEQ IDNO:2 promoter- CTACCTGTGTCGGTTTGCGGT primer non-T7GCGAAGTTTAGTAGCGAAGTTAGTGATGT SEQ ID NO:3 primer ProbeGCUUCUAGCGAUACAUAUACUCUACCC SEQ ID NO:4

The results presented in FIG. 5 confirmed that the sample preparationprocedure which included an alkaline shock yielded dramatically improvedresults over standard procedures. In every case, samples were judged aspositive if the chemiluminescent signal indicating detection of rRNAamplicons exceeded the signal detected in control trials conducted using1000 copies of the rRNA template. As indicated in the figure, trialsconducted using at least 20 GBS bacteria as the source of nucleic acidtemplates uniformly gave positive results, regardless of whether thealkaline shock was included in the sample preparation procedure.However, while the standard sample preparation procedure was useful forreliably detecting as few as about 10 GBS bacteria when used inconjunction with an in vitro amplification and detection assay, theprocedure that included the alkaline shock could be used for reliablydetecting as few as a single bacterium. These conclusions are based on astatistical analysis, where, among a collection of replicate samplesreceiving an aliquot intended to contain one bacterium some samples willcontain none and some samples will contain two bacteria. Clearly, thealkaline shock sample preparation procedure dramatically improveddetection of the bacterial target nucleic acid.

This invention has been described with reference to a number of specificexamples and embodiments thereof. Of course, a number of differentembodiments of the present invention will suggest themselves to thosehaving ordinary skill in the art upon review of the foregoing detaileddescription. Thus, the true scope of the present invention is to bedetermined upon reference to the appended claims.

1. A method of processing a biological sample, comprising the steps of:(a) combining said biological sample with a pH buffer and a detergentthat lyses or disrupts biological membranes that may be present in saidbiological sample, whereby there is created a first liquid compositionhaving a first pH; (b) mixing with said first liquid composition analkaline composition, whereby there is created a second liquidcomposition having a second pH, wherein said alkaline composition is anaqueous solution comprising a strong base at a concentration of at least0.1 N, wherein said second pH is at least 0.2 pH units higher than saidfirst pH, and wherein said second pH is lower than pH 9.5; and (c)purifying nucleic acids from said second liquid composition.
 2. Themethod of claim 1, wherein purifying step (c) comprises, (i)immobilizing nucleic acids from said second liquid composition onto asolid support by sequence-specific target capture; and (ii) washing thesolid support to remove any material not immobilized thereon.
 3. Themethod of claim 1, wherein purifying step (c) comprises, (i)immobilizing nucleic acids from said second liquid composition onto asolid support by non-specific target capture that is substantiallyindependent of base sequence; and (ii) washing the solid support toremove any material not immobilized thereon.
 4. The method of claim 1,wherein purifying step (c) comprises immobilizing onto a solid supportnucleic acids from said second liquid composition, and then washing thesolid support to remove any material not immobilized thereon.
 5. Themethod of claim 4, wherein the step for immobilizing onto the solidsupport comprises immobilizing by sequence-specific target capture. 6.The method of claim 4, wherein the step for immobilizing onto the solidsupport comprises immobilizing by non-specific target capture that issubstantially independent of base sequence.
 7. The method of claim 4,wherein the nucleic acids purified in step (c) comprise RNA.
 8. Themethod of claim 4, wherein the nucleic acids purified in step (c)comprise RNA and DNA.
 9. The method of claim 4, wherein the nucleicacids purified in step (c) comprise DNA.
 10. The method of claim 4,further comprising the step of performing an in vitro nucleic acidamplification reaction using the nucleic acids purified in step (c) astemplates.
 11. The method of claim 10, wherein said in vitro nucleicacid amplification reaction comprises a reverse transcriptase.
 12. Themethod of claim 10, wherein said in vitro nucleic acid amplificationreaction is a multiplex reaction.
 13. The method of claim 10, whereinthe in vitro nucleic acid amplification reaction is an isothermal invitro nucleic acid amplification reaction.
 14. The method of claim 4,wherein said solid support comprises paramagnetic monodisperseparticles.
 15. The method of claim 14, further comprising the step ofperforming an in vitro nucleic acid amplification reaction using thenucleic acids purified in step (c) as templates.
 16. The method of claim15, wherein said in vitro nucleic acid amplification reaction comprisesa reverse transcriptase.
 17. The method of claim 15, wherein said invitro nucleic acid amplification reaction is a multiplex reaction. 18.The method of claim 15, wherein the in vitro nucleic acid amplificationreaction is an isothermal in vitro nucleic acid amplification reaction.19. The method of claim 4, wherein said second pH is in the range offrom pH 8.0 to 9.2.
 20. The method of claim 19, wherein said strong baseis selected from the group consisting of NaOH and LiOH.
 21. The methodof claim 1, further comprising the step of performing an in vitronucleic acid amplification reaction using the nucleic acids purified instep (c) as templates.
 22. The method of claim 21, wherein said in vitronucleic acid amplification reaction comprises a reverse transcriptase.23. The method of claim 21, wherein said in vitro nucleic acidamplification reaction is a multiplex reaction.
 24. The method of claim21, wherein the in vitro nucleic acid amplification reaction is anisothermal in vitro nucleic acid amplification reaction.
 25. The methodof claim 21, wherein the nucleic acids purified in step (c) compriseRNA.
 26. The method of claim 21, wherein the nucleic acids purified instep (c) comprise RNA and DNA.
 27. The method of claim 21, wherein thenucleic acids purified in step (c) comprise DNA.